In modern medicine, immunotherapy or vaccination has virtually eradicated diseases such as polio, tetanus, tuberculosis, chicken pox, measles, hepatitis, etc. The approach using vaccinations has exploited the ability of the immune system to prevent infectious diseases. Vaccination with non-live materials such as proteins generally leads to an antibody response or CD4+ helper T cell response. (Raychaudhuri and Morrow (1993) Immunology Today 14:344). On the other hand, vaccination or infection with live materials such as live cells or infectious viruses generally leads to a CD8+ cytotoxic T-lymphocyte (CTL) response. A CTL response is crucial for protection against cancers, infectious viruses and certain bacteria. This poses a practical problem, for, the only way to achieve a CTL response is to use live agents which are themselves pathogenic. The problem is generally circumvented by using attenuated viral and bacterial strains or by killing whole cells which can be used for vaccination. These strategies have worked well but the use of attenuated strains always carries the risk that the attenuated agent may recombine genetically with host DNA and turn into a virulent strain. Thus, there is need for methods which can lead to CD8+ CTL response by vaccination with non-live materials such as proteins in a specific manner. It has been discovered that heat shock protein complexes have particular utility as vaccines against cancers and infectious diseases. (Srivastava et al., (1994) Curr. Op. Immu. 6:728; Srivastava (1993) Adv. Cancer Res. 62:153).
Chaperone proteins are involved in a wide array of events involving the processing and functioning of cellular proteins. Chaperone proteins were originally recognized for their protective role during cell stress. It is now clear that chaperone proteins also are involved in folding, unfolding, refolding, stabilizing, oligomerizing, salvaging, and discarding cellular proteins during the routine events of intracellular activities. Chaperone proteins perform these functions as multi-protein complexes consisting of chaperones, co-chaperones, and substrate molecules.
Heat shock proteins (HSPs) are proteins synthesized by a cell in response to heat shock. There are also some HSPs that are homologous to stress induced proteins and are expressed constitutively. HSPs can include a protein that (i) increases in concentration when a cell is exposed to a stressful stimulus; (ii) binds other proteins or peptides; and (iii) is capable of releasing the bound proteins or peptides in the presence of adenosine triphosphate (ATP) or low pH. An HSP can also be any protein or conserved homolog thereof whose intracellular concentration does not increase when a cell is exposed to stressful stimulus and that shows at least 35% homology with a known HSP protein as determined by the BLAST p algorithm. HSPs are a type of chaperone protein. Heat shock proteins fall into families such as but not limited to the families HSP25/HSP27, HSP60, HSP70 and HSP90 where the numbers reflect the approximate molecular weight of the stress proteins in kilodaltons. Heat shock proteins are capable of binding proteins or peptides, with which they form complexes endogenously in cells or in vitro under the appropriate conditions (Nair et al. (1999) J. Immun. 162:6426; Flynn et al. (1989) Science 245:385; Blachere et al. (1997) J. Exp. Med. 186:1315).
Immunization of mice with the gp96 complex or hsp84/86 complex isolated from a particular tumor rendered the mice immune to that particular tumor, but not to antigenically distinct tumors. Isolation and characterization of genes encoding gp96 and hsp84/86 revealed significant homology between them, and showed that gp96 and hsp84/86 were, respectively, the endoplasmic reticular and cytosolic counterparts of the same heat shock proteins (Srivastava, et al. (1988) Immunogenetics 28:205; Srivastava, et al. (1991) Curr. Top. Microbiol. Immunol 167:109). Further, hsp70 complex was shown to elicit immunity to the tumor from which it was isolated but not to antigenically distinct tumors. However, hsp70 complex depleted of peptides was found to lose its immunogenic activity (Udono and Srivastava (1993) J. Exp. Med. 178:1391). The observations revealed that the heat shock proteins are not immunogenic per se, but are carriers of antigenic peptides that elicit specific immunity to cancers (Srivastava (1993) Adv. Cancer Res. 62:153).
This phenomenon has been observed in both tumor and viral models with known and unknown antigens (Srivastava, et al. (1998) Immunity 8:657; Ciupitu, et al. (1998) J. Exp. Med 5: 685; Arnold et al. (1995) J. Exp. Med. 182:885). The presence of an antigenic peptide bound to gp96, hsc70, and hsp84/hsp86 has been structurally demonstrated in cells for which the antigenic peptide is known (Nieland, et al. (1996) Proc. Nat'l. Acad. Sci. USA 93:6135; Breloer, et al. (1998) Eur. J. Immunol. 28:1016; Ishii et al. (1999) J. Immunol. 162:1303). Vaccination with heat shock protein complexes is applicable for both the prophylactic (Srivatava, et al. (1986) Proc. Nat'l. Acad. Sci. USA 83:3407; Ullrich, et al. (1986) Proc. Nat'l. Acad. Sci. USA 83:3121; Peng, et al. (1997) J. I. Meth. 204:13; Basu and Srivastava (1999) J. Exp. Med. 189:797) and therapeutic treatment of cancer (Tamura et al. (1997) Science 278:117; Yedavelli, et al. (1999) Int. J. Mol. Med 3:243) and for the treatment of infectious diseases (Ciupitu et al. (1998) J. Exp. Med. 5:685). The translation of this approach to immunotherapy of human cancer is currently under investigation using either gp96 complex (Janetzki, et al.(2000) Int. J. of Cancer 88:232; Amato, et al. (1999) ASCO meeting, abstract 1278; Lewis, et al. (1999) ASCO meeting abstract 1687) or hsp70 complex as an autologous vaccine and the individual patient's cancers as a source of the heat shock proteins (Ménoret and Chandawarkar (1998) Semin. in Oncology 25:654).
The preparation and use of a customized, autologous vaccine against the tumors of individual patients is now feasible using tumor-derived hsp complexes (Ménoret and Chandawarkar (1998) Semin. in Oncol. 25:654). Preliminary clinical trials with this approach have demonstrated that patients immunized with gp96 complex, purified from their own tumors, develop cancer-specific CD8+ T cell response (Janetzki, et al.(2000) Int. J. of Cancer 88:232; Lewis, et al. (1999) ASCO meeting abstract 1687; Amato, et al. (1999) ASCO meeting abstract 1278). Clinical trials using purified preparations of autologous tumor-derived hsp70 complex are being conducted for the treatment of breast carcinoma and chronic low grade leukemia. Preliminary evidence of clinical responses to purified gp96 complex has also been obtained in a trial with patients with renal carcinoma (Amato, et al. (1999) ASCO meeting abstract 1278). These trials rely on a time-tested immunologic principle that anti-tumor immunity is generally private (Srivastava, et al. (1998) Immunity 8:657; Berd, et al. (1999) Semin. Oncol. 25:1315), justifying the use of a patient's own tumor as the source of the anti-tumor vaccine.
Previous methods for purification of the immunogenic chaperone protein complexes led to the isolation of chaperone protein species (U.S. Pat. Nos. 5,997,873; 5,935,576; 5,750,119; 5,961,979; 5,837,251). These methods neglected the rest of the sample that could be used as a complementary source of chaperone protein-based vaccine. Therefore, it is an object of the invention to provide a method for collecting multiple chaperone proteins and chaperone protein complexes from a limited sample source.
Citation or identification of any reference herein shall not be construed as an admission that such reference is available as prior art to the present invention.